Volume regulation is a feature common to many vertebrate cells. When placed in hypotonic bathing solution, fluid transporting epithelial cells, peripheral blood cells and even glia and neurons initially swell, but then, over several minutes, shrink back to near their resting volumes. The regulatory volume decrease (RVD) is usually accompanied by the passive loss of intracellular K+ and anions including Cl-. Recently, using cell-attached patch clamp recording, we have investigated ion channel activity during cell swelling and RVD in clonal N1E115 neuroblastoma cells. The activity of a stretch-sensitive, non-selective cation channel (C+(SA)) increases shortly after the onset of osmotically induced cell swelling. Shortly thereafter, and roughly coincident with the onset of RVD, two types of voltage-dependent channels spontaneously open at the resting potential: (1) a delayed-rectifier type K+ channel, and (2) large conductance anion channel. We have hypothesized that the C+ (SA) channel may be a volume "sensor" mechanism, while the voltage- dependent K+ and anion channels may be potassium salt exit pathways during RVD. We now propose to test the latter hypothesis by combining videomicroscopic imaging of cell size with (a) single-channel recording and (b) the perforated patch variant of whole-cell recording to examine changes in membrane current, voltage and resistance during cell swelling and RVD in neuroblastoma under a variety of osmotic perturbations, and in the presence and absence of ion channel blockers which we shall demonstrate to be selective for a given channel. We shall focus on ascertaining (a) when and how many each ion channel type opens during RVD; (b) what forces and/or intracellular messengers actually gate each channel, as well as which ions permeate each channel when open; and (c) whether the net sum ion efflux through these channel during the RVD accounts for much or all of the salt loss from the cell. These results may help to elucidate the mechanism used by brain cells to limit swelling during hyponatremia, ischemia, insult by neurotoxic excitatory transmitters, and even cell growth.